Method for making fluorinated sugars having a side chain and use thereof

ABSTRACT

Furanosides or pyranosides can be reacted with pyruvate and converted into sialic acids in good yields by an enzymatic synthesis, using a suitable aldolase, where the concentration of aldolase, per 50 mM of pyruvate, is 1 to 2,500 U*/ml. For example, 3-fluoroneuraminic acid and other 3,3-didesoxy-3-fluorononulopyranosonic acid derivatives can be prepared in this manner. The F-atom in the resulting sialic acid (which can be  19 F or  19 F and  18 F) provides a label which makes possible studies of mammalian physiology and diagnosis of mammalian diseases.

This application is a continuation-in-part of U.S. application Ser. No.09/156,322, filed Sep. 18, 1998, now abandoned.

FIELD OF THE INVENTION

This invention relates to the enzymatic synthesis of fluorinated sugars(including fluorinated amino or amido sugars) which have a cyclicstructure and a side chain and hence have a nucleus of more than 6carbon atoms. An aspect of this invention relates to the synthesis offluorinated sialic acids or fluorinated nonulosaminic acids, a family ofamino sugars containing at least 8, more typically at least 9, carbonatoms.

Another aspect of this invention relates to the enzymatic rid synthesisof 3-fluoroneuraminic acid (5-acetamido-3,5-didesoxy-3-fluoro-D-glycero-D-galacto-nonulopyranosonic acid) andother 3,5-didesoxy-3-fluorononulopyranosonic acid derivatives and theiruse.

DESCRIPTION OF THE PRIOR ART

The sialic acids are generally cyclic pentoses (furanoses) or cyclichexoses (pyranoses) with a side chain (typically three carbon atomslong); accordingly, the sugar nucleus of these compounds has 8 or 9carbon atoms, and this nucleus can be substituted with an amino groupwhich can be in turn be substituted with acyl groups and the like. Themost important embodiments of this class of compounds can be consideredto be derivatives of amino sugars such as neuraminic acid, C₉H₁₇NO₈. Atleast five sialic acids occur in nature; they are widely distributedthroughout the animal kingdom (including some bacteria as well as morecomplex organisms such as mammals) and appear to be regular componentsof glycoproteins and glycolipids (where they typically occupy terminalpositions). The most important of the sialic acids is generallyconsidered to be the N-acetyl derivative of neuraminic acid (“Neu5Ac”),i.e. 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid.

Preparations of sialic acids having 8- as well as 9-carbon nuclei aredescribed in the scientific literature. For an example of a synthesisinvolving a furanose such as D-arabinose, see U. Kragl et al, J. Chem.Soc. Perkin. Trans. 1:119-124 (1994).

Because Neu5Ac and similar sugars occur so widely in higher animals andare so intimately involved in the physiology of mammals, they can beused as diagnostic and investigative tools, provided that they aresuitably labeled with an isotope which can be measured or detected byvarious imaging techniques, radioactivity measurements, and othernon-invasive procedures.

Fluorine is one of the most important labeling elements. The stableisotope, ¹⁹F, provides opportunities for in vivo investigation with¹⁹F-NMR spectroscopy and has advantages over ¹H-NMR spectroscopy used inMR-tomography. In the body, fluorine occurs naturally only in teeth andbones. Therefore, it is possible to observe the kinetics of thebiodistribution of fluorine-labeled compounds.

Fluorine has several isotopes in addition to ¹⁹F, all of which areunstable, but only one of these has practical significance: the isotope,¹⁸F, which is radioactive and has the longest half-life of the unstableisotopes (the other unstable isotopes have half-lives lasting less than3 minutes). The ¹8F isotope has a half-life of 110 minutes and is veryuseful in biological studies and in medicine, but a half-life of lessthan 2 hours does impose some limits on its utility. Examples of theuses of ¹⁸F include non-invasive measurement of pharmacokineticphenomena and the localization of tumors with ¹⁸F-labeled2-fluorodesoxyglucose (e.g. by positron emission tomography).

The short half-life of the ¹⁸F isotope can impose severe requirementsupon methods for synthesizing the ¹⁸F-labeled compound. The yield oflabeled compound should be high, and, even more important, the synthesismust be very rapid.

In any method in which an F-labeled compound is used, it is generallynecessary that its physiological properties (e.g. its properties as asubstrate for an enzyme) be similar to the endogenous, non-fluorinatedcompound it is supposed to mimic. The fluorine atom has the advantage ofbeing fairly small in its covalent radius and hence does not differ toomarkedly from hydrogen in terms of steric hindrance. A fluorinesubstituent does differ from other substituents in terms of chargedensity, due to its high electronegativity and electronic density. Butgenerally speaking, the advantages of fluorine as a labeling substituentfar outweigh its disadvantages.

A number of F-labeled derivatives of Neu5Ac are known. For example, the3-fluorine derivative (“Neu5Ac3F”), which has the systemic name5-acetamido-3,5-didesoxy-3-fluoro-D-glycero-D-galacto-nonulopyranosonicacid, can be prepared by the aldol condensation of N-acetylmannosamine(“ManNAc”) and fluoropyruvate or β-fluoropyruvic acid (F—CH₂—CO—COOH,systemic name 1-fluoro-2-oxopropanoic acid). The yield, however, ismoderate (1.5%) and the purification is labor intensive. A recent paperdiscloses an electrophilic selective fluoridation which provided yieldsup to 80% and diastereomer selectivity of 75%. But the need for a fasterand more stereospecific synthesis still exists.

The biosynthesis of Neu5Ac is enzyme-catalyzed and would appear toprovide a model for a quick synthesis. In the biosynthesis, ManNAc isreacted with the pyruvate (pyruvic acid, CH₃—CO—COOH), and the enzymecatalyst is N-acetylneuraminic acid aldolase. N-acetylneuraminic acidaldolase EC 4.1.3.3 can be found in animal tissue and some bacteria.This enzyme has also been produced by biotechnology methods involvingcommon microorganisms such as E. coli. The natural sialic acid Neu5Achas been made successfully by enzymatic synthesis, but the preparationof Neu5Ac derivatives (particularly Neu5Ac3F) is more problematic.

The enzymatic synthesis of Neu5Ac3F has been investigated. For example,the amounts of substrates (the sugar component, ManNAc, and thepyruvate, β-fluoropyruvate) have been varied considerably. The ManNAcwould be expected to react with the β-fluoropyruvate (“F-pyr”) to formNeu5Ac F-substituted at the 3-position (Neu5Ac3F), but under theconditions chosen in the If literature (50 mM of both substrates, 0.1U*/ml and N-acetylneuraminic acid aldolase [“Neu5Ac-aldolase”] from E.coli in water, buffered at a pH of 7.7, maintained at 37° C. andincubated for 24 hours), no conversion to Neu5Ac3F was observed. (Theexpression U*, a convenient enzyme activity unit employed in presentingdata hereafter, was devised by the enzyme supplier, Toyobo, and is theenzyme activity with respect to the standard reaction of “Pyr”(pyruvate)+ManNAc to obtain Neu5Ac. The expression U [without theasterisk], also used hereafter, is the enzyme activity with respect tothe reaction of F-Pyr+ManNAc to Neu5Ac3F.)

Accordingly, further investigation of the enzymatic synthesis of thesefluorinated sugars (particularly fluorinated amino sugars such asNeu5Ac3F) is needed.

SUMMARY OF THE INVENTION

It has now been discovered that the manipulation of enzyme synthesisconditions can provide a high-yield and preferably rapid preparation offluorinated ≧8-carbon sugars from the appropriate substrates using analdolase enzyme. First and foremost, the enzyme concentration must beincreased drastically in comparison to the concentration reported in theliterature. This drastic increase in enzyme concentration does notcreate any serious economic drawback. The enzyme obtained throughbiotechnology methods, e.g. from E. Coli, is relatively inexpensive. Theenzyme Neu5Ac-aldolase from C. perfringens is also commerciallyavailable and can be used in this invention with results similar to theenzyme from E. coli. Moreover, the enzyme exhibits remarkable stabilityand can be recovered and reused in subsequent cycles or batches.

To insure that the synthesis will be fast enough to permit the use of¹⁸F-labeled compounds, the sugar component is preferably present, inmolar terms, in a large excess by comparison to the fluorinatedcomponent. The fluorinated component concentration should be sufficientalso, however, and it is preferred, in the synthesis of ¹⁸F-labeledcompounds, to introduce a significant amount of ¹⁹F-containing substrateas a carrier.

For cost-effectiveness, the synthesis is carried out on a scale in whichthe amounts of substrates are in the tens or hundreds of millimoles.Aldolase enzyme activity peaks within the range of 30-60 mM of F-pyr andgenerally increases with increasing amounts (e.g. >300, preferably >400mM) of the sugar. The aldolase enzyme concentration is preferably inexcess of 1 U*/ml and can range as high as 2,500 U*/ml. When the amountof F-Pyr is within the desired range (e.g. about 50 mM), the preferredaldolase enzyme concentration is 20 to 500 U*/ml, 100 to 200 U*/ml beingespecially preferred. Further, the reaction can also depend ontemperature and pH. The preferred temperature range from about 1° C. toabout 55° C. The preferred pH can range from about 5 to about 10.

BRIEF DESCRIPTION OF THE DRAWING

The principles of this invention are illustrated by the accompanyingDrawings, wherein the results of several kinetic studies are showngraphically. The first three kinetic studies were carried out with 24U*/ml (24 U*/mg enzyme lyophilisate). The enzyme activity was measuredas a function of the substrate concentrations and the productconcentration.

Thus, FIG. 1A is a plot of enzyme activity as a function of thefluoropyruvate (F-pyr) concentration (in mM), under the followingconditions: 24 U*/ml (1 mg/ml) of the aldolase enzyme; 300 mM of theother substrate, N-acetylmannosamine (ManNAc); a pH of 7.5; and atemperature of 25° C.

FIG. 1B is a plot of enzyme activity as a function of the ManNAcconcentration (in mM); in this case the F-pyr substrate is present at aconcentration of 50 mM; the amount of aldolase is 24 U*/ml (1 mg/ml);and the pH and temperature are again 7.5 and 25° C., respectively.

FIG. 1C is a plot of enzyme activity as a function of Neu5Ac3Fconcentration in mM; the aldolase was again 24 U*/ml (1 mg/ml); theManNAc substrate concentration was 300 mM; the F-pyr substrateconcentration was 50 mM; the pH was 7.5; and the temperature was 25° C.

Since Neu5Ac3F can exist in the form of stereoisomers, and the aldolaseproduces two diastereomers (though not in equal amounts, the relativeamounts being dependent upon reaction conditions), an additional studywas done to investigate the effect of the reaction conditions on therelative amounts of the two diastereomers.

Thus, FIG. 2 is a plot of diastereometric excess (excess of thediastereomer favored by the enzyme synthesis) and equilibrium conversionas a function of the total concentration (in mM), i.e. ManNAc+Neu5Ac3Fwith equal concentrations of substrates (F-pyr=ManNAc); the equilibriumconstant (K_(eq))=8500±5600 l/mol.

As noted previously, the aldolase enzyme is reasonably stable and can berecovered and reused.

Thus, FIG. 3 illustrates the performance (% conversion vs. time inhours) of repetitive batches where the substrate concentrations wereequal (ManNAc=F-pyr=200 mM), but the aldolase was much higher (192 U*/ml(8 mg/ml); pH=7.5; temperature=25° C. Enzyme had to be added tocompensate for deactivation (33%), and the diastereometric excess was91%.

FIG. 4 illustrates a rapid conversion of [¹⁸F]F-Pyr tracer with[¹⁹F]F-Pyr and ManNAc to Neu5Ac3F (mM of Neu5Ac3F produced vs. time inminutes), where the ManNAc substrate is in large excess (125 mM)compared to the F-pyr (1 mM [¹⁹F]F-Pyr with 100-300 μCi of [¹⁸F]F-Pyr).Other conditions: aldolase=125 U/ml (5 mg/ml), pH=7.5, andtemperature=25° C. The equilibrium conversion was >99%.

FIG. 5 illustrates enzyme activity as a function of temperature withpH=7.5, [ManNAc]=400 mM, [F-Pyr]=50 mM and Aldolase=1 mg/ml.

DETAILED DESCRIPTION

In the case of the preferred pyranose starting materials, the reactionwhich is facilitated to a surprising degree by this invention can berepresented in general terms as Reaction 1 (“Rx 1”):

where R is OH or a nitrogen-containing group such as amino or amido, andM⁺ is H⁺ or a pharmaceutically-acceptable cation.

As indicated above, substrate (I) is a sugar or amino sugar typicallyhaving a Haworth-Hirst pyranoside nucleus. However, substrate (I) canalso be a furanoside. Substrate (II) is the fluoropyruvate (F-pyr), andproduct (III) has a fluorine label and a nine-carbon nucleus. The Rgroup can increase the total number of carbons. For example, if R is—NH—CO—CH₃, the total number of carbons in the product would be 11.

Although amino sugars are preferred as substrate (I), mannose (a hexosein which R=OH) has been successfully converted with F-pyr in accordancewith this invention to obtain ketodesoxy-nonulopyranosonic acid(“F-KDN”).

Pyranosides and compounds derived from them such as sialic acidsgenerally have rings with the “chair” configuration of cyclohexane, andlike cyclohexane, the rings can have axial and equatorial substituents.In the case in which substrate (I) is ManNAc but substrate (II) ispyruvic acid, the resulting product (Neu5Ac) has no stereoisomerism atthe 3-position. But when F-pyr is substrate (II), the two substituentsat the 3-position are not the same, and the F-substituent can beoriented either axially or equatorially. This steric effect isillustrated by Reaction 2 (“Rx 2”), in which substrate (I) is ManNAc(shown in the chair configuration) and substrate (II) is F-pyr; “Ac”represents the acetyl group, CH₃CO—, hence -NHAc is the acetamido group,—NH—CO-—H₃:

When Rx 2 is carried out under the conditions employed in thisinvention, the amount of diastereomer with the fluorine substituent inthe axial position (“axial-F diastereomer”) far exceeds the diastereomerwith the equatorial fluorine substituent (“equatorial-F diastereomer”).The ratio of axial-F:equatorial-F is influenced by the reactionconditions (total concentration, substrate ratio, enzyme concentration).Via ¹⁹F-NMR spectroscopy, it can be shown that the axial-F diastereomeris formed in excess at a total of concentration of >3 mM.

The axial diastereomer, which is the main product and is mixed with arelatively small amount of the byproduct (the equatorial-F diastereomer)can be purified by methods well known to those skilled in the art, e.g.by elution from an ion exchange medium.

FIG. 2 shows the dependence of the equilibrium position (% ofequilibrium conversion) and the diastereomeric excess upon totalconcentration ([ManNAc]+[Neu5Ac3], with [F-pyr]=[ManNAc]).

As noted previously, equality of concentration of the two substrates isnot necessarily ideal, particularly if a rapid synthesis is desired.FIG. 1A illustrates a kinetic study carried out at various F-pyrconcentrations with the ManNAc concentration set at 300 mM, and FIG. 1Billustrates the effect of ManNAc concentration when the F-pyrconcentration is set at 50 mM. FIGS. 1A and 1B (and cf. FIG. 4) suggestthat the concentrations of each substrate are important variablesaffecting enzyme activity. The kinetic study in FIG. 1A is considered todemonstrate that a concentration of 30-60, preferably 40-50, mM F-Pyrleads to the highest enzyme activity. FIG. 1B is considered to show thatthe enzyme activity also increases with increasing ManNAc concentration,and a ManNAc concentration greater than 300 mM, preferably >400 mM, isdesirable.

Enzyme kinetics is a very useful tool for providing insights intoenzyme-catalyzed reaction mechanisms. The simplest case, where a freeenzyme and a single substrate form an enzyme-substrate complex whichreverts back to free enzyme+substrate and/or forms products, can bedescribed in an equation which relates the velocity of the reaction (v)to total enzyme concentration (free enzyme concentration+concentrationof complexed enzyme), substrate concentration, rate constants, and theMichaelis constant (K_(m)). This approach has been successfully extendedto more complicated enzyme-catalyzed reactions in which more than onesubstrate is present. Using an equation that describes—in the “standard”ManNAc/Pyr reaction—the dependence of the reaction velocity (and hencethe enzyme activity) upon the substrate concentrations c_((pyr)) andc_((ManNAc)) and the product concentration c_((Neu5Ac)), an equation canbe modeled for reaction velocity or enzyme activity in the seeminglyanalogous fluorine-labeling reaction between ManNAc and F-pyr. Theresulting model kinetic equation (“Equation 1”) is set forthsubsequently. Like the equation for the standard, ManNAc/Pyr reaction,the concentration value c_((ManNAc)) is used, but the concentrationvalues of the other substrate, i.e. _(c(F-pyr)), and the product,c_((Neu5Ac3F)), are taken from the ManNAc/F-pyr reaction. The kineticparameters of Equation 1 were determined from data measured underinitial reaction conditions by means of non-linear regression. Among thekinetic parameters of the model equation are the K_(m) values for F-pyrand ManNAc. The K_(m) value for ManNAc (393 mM) matches well with thevalue given in the literature for the standard, ManNAc/Pyr reaction (402mM). The K_(m) value for F-pyr (41 mM) is about 5 times higher than theone for the standard reaction (8.5 mM) and additionally a substratesurplus inhibition is observed.

Equation 1 is given below:

V=(a*b*v_(max))/((a+(a²/K_(ia))+(K_(ma)*(1+(P/K_(ip)))))*(b+K_(mb)*(1+(P/K_(ip)))))

where:

V_(max) = 0.898 ± 0.347 U/mg a = c_((F-Pyr)) mM K_(ma) = 41 ± 20 mMK_(ia) = 49 ± 25 mM b = c_((ManNAc)) mM K_(mb) = 393 ± 168 mM P =C_((Neu5Ac3F)) mM K_(ip) = 644 ± 123 mM,

and where the kinetic parameters referred to above are V_(max) (maximalreaction rate), K_(ma) (K_(m)-value of F-Pyr), K_(mb) (K_(m)-value ofManNAc), K_(ia) (substrate surplus inhibitor constant for F-pyr) andK_(ip) (product inhibitor constant of Neu5Ac3F). In the foregoingequation, an asterisk is used to indicate a product (i.e. x*y means theproduct of x and y).

The differences between the standard, ManNAc/Pyr reaction and theManNAc/F-pyr reaction have important consequences. While the maximalactivity of the standard reaction reaches 13.8 U/mg (with an enzymecharge of >15 U*/mg), the maximal activity of the reaction with F-pyr isreduced to 0.9 U/mg (with an enzyme charge of >24 U*/mg). This is anexplanation for the observation in the literature (Y. Uchita et al., J.Biochem. 96: 507-514 [1984]) that no conversion took place. Theconcentration of 50 mM F-pyr is well chosen in view of the K_(m)-valueand the substrate surplus inhibition of F-pyr, but the 50 mMconcentration of ManNAc is far below the mi-value of 393 mM. At the 50mM concentration, only an activity of 0.035 U/mg of enzyme (with 24U*/mg) is reached, which corresponds to 1/685 part of the applied enzymeactivity. With a standard activity of 0.1 U*/ml this would correspond toan activity of 1.46*10⁻⁴ U/ml. With the definition for one unit, 1 U=1μmol/min; this results in a product formation of 1.46*10⁻⁴μmol*(min*ml)³¹ ¹ or 0.21 μmol*(d*ml)³¹ ¹ (see the foregoing 5description of the use of the asterisk to indicate products).

FIG. 1B confirms that enzyme activity increases with increasing(ManNAc):(F-pyr) and that the ManNAc concentration should be well inexcess of 50 mM. FIG. 1A confirms that 50 mM is a good choice for theconcentration of the F-pyr substrate.

Some loss of enzyme activity occurs as the product concentrationincreases (see FIG. 1C), but enzyme activity losses can be compensatedfor.

Although F-pyr is typically F—CH₂—CO—COOH, the synthesis of thisinvention can take place under mildly basic conditions, under whichF-pyr can be F—CH₂—COO^(31 M+), where M⁺is H⁺and/or a pharmaceuticallyacceptable cation such as an alkali metal cation.

The invention is further illustrated by the following non-limitingExamples.

EXAMPLE 1 Recovery and Reuse of Enzyme in Repeated Cycles

The substrates ManNAc and F-pyr were incubated at a concentration of 200mM and an enzyme concentration of 8 mg/ml (192 U*/ml) at 25° C. and pH7.5. After the reaction was completed, the solution was centrifuged inan Amicon Centriprep under retention of the enzyme by an ultrafiltrationmembrane. Thereafter, a new substrate solution was added to the enzyme.Due to the loss of activity by the filtration step, 4 mg/ml enzyme wereadded every 3 cycles.

From 6 cycles, 2 g product were isolated with a diastereomeric excess of91%. The course of the two first cycles is shown in FIG. 3. Thepurification of the main-product (F-axial) was achieved with an anionexchanger (Dowex 1X2 formate form, elution with formic acid gradient 0→1molar).

EXAMPLE 2 Labeling With ¹⁸F (Rapid Synthesis)

Neu5Ac3[¹⁸F]F was produced by an enzyme synthesis using the substrates[¹⁸F]F-pyr and ManNAc in the presence of [¹⁹F]F-pyr as a carrier.Because of the need of a fast conversion in the enzyme reaction due tothe short half-life of the ¹⁸F, a sufficient substrate concentration isnecessary. Therefore 1 MM a [¹⁹F]F-pyr was added to the [¹⁸F]F-pyr ascarrier. For the same reason, ManNAc was applied in a high excess of 125mM. The assigned radioactivity was 100-300 μCi which corresponds to aradioactivity concentration of nano- to picomolar. The enzymeconcentration was 5 mg/ml. By using the kinetic model (Equation 1) theconversion time course shown in FIG. 4 could be simulated. Aquantitative measurement was not possible with the available analytictechnique, because of an inadequate baseline.

EXAMPLE 3 Mannose And F-pyr As Substrates

Mannose was also successfully converted with F-pyr to F-KDN(ketodesoxynonulo-pyranosonic acid). Again both epimers are formed indifferent amounts (axial epimer >95%). The reaction is slower than theManNAc/F-pyr reaction and the conversion attained is smaller. Thequantification was difficult, however, due to the poor UV activity ofboth substrates and the product. Under the same conditions as in Example1, in two repetitive reaction cycles, 500 mg product were synthesized.

COMPARATIVE EXAMPLES

A. (ManNAc)=(F-pyr)=50 mM, And Low Enzyme Activity

The work of Y. Uchita et al. was repeated under the conditions reportedin that paper, and the same results were observed. Measurable conversionto Neu5Ac3F was not observed.

B. ManNAc+β-Hyroxypyruvate

An attempt was made to substitute β-hydroxypyruvate for F-pyr. Theexperiment showed that a conversion of β-hydroxypyruvate (unlike F-pyr)takes place only to a very small extent. When interacting with Neu5Ac,the hydroxy radical differs significantly from hydrogen not only inelectron density but in size as well. Apparently the steric factor playsa much larger role in this reaction than in the ManNAc/F-pyr reaction.

C. ManNAc+β-Bromopyruvate

The attempt to substitute bromopyruvate for fluoropyruvate was notsuccessful. Conversion of bromopyruvate was not observed.

We claim:
 1. An enzymatic synthesis of a fluorinated sugar having atleast 8 carbon atoms, said synthesis comprising reacting aF—CH₂—CO—COO⁻M⁺ with a furanoside or pyranoside sugar substrate in thepresence of an aldolase enzyme, wherein M⁺ is H⁺ or a pharmaceuticallyacceptable cation, and wherein the concentration of aldolase enzyme, per50 mM of F—CH₂—CO—COO⁻M⁺, is 1 to 2,500 U*/ml, where U* represents anenzyme activity unit and is the enzyme activity with respect to thestandard reaction of pyruvate and N-acetylmannosamine to obtain theN-acetyl derivative of neuraminic acid, further comprising the step ofrecovering the fluorinated sugar.
 2. The synthesis according to claim 1,wherein the amount of the fluorinated sugar produced is sufficient tostudy mammalian physiology or diagnose mammalian diseases.
 3. Anenzymatic synthesis according to claim 1 wherein said reaction isrepresented by the following equation:

wherein R is OH or a nitrogen-containing group, and M⁺ is apharmaceutically-acceptable cation.
 4. An enzymatic synthesis accordingto claim 1, wherein the sugar substrate is an amino or amido sugar, andthe resulting product is a fluorinated sialic acid.
 5. An enzymaticsynthesis according to claim 4, wherein said enzyme concentration isabout 20 to about 500 U*/ml.
 6. An enzymatic synthesis according toclaim 1, wherein the sugar substrate is N-acetylmannosamine.
 7. Anenzymatic synthesis according to claim 1, wherein the sugar substrate ismannose.
 8. An enzymatic synthesis according to claim 5, wherein thesugar substrate is N-acetylmannosamine.
 9. An enzymatic synthesisaccording to claim 1, wherein the aldolase enzyme is N-acetylneuraminicacid aldolase E.C. 4.1.3.3.
 10. An enzymatic synthesis according toclaim 1, wherein the synthesis is a batch process wherein activealdolase enzyme is recovered and reused in subsequent batches.
 11. Anenzymatic synthesis according to claim 1, wherein the concentration ofaldolase enzyme is 100 to 200 U*/ml.
 12. An enzymatic synthesisaccording to claim 1, wherein the reaction medium provides an enzymeactivity of at least about 0.02 U/ml, where U represents the enzymeactivity with respect to the reaction of said substrates F—CH₂—CO—COO⁻M⁺and N-acetylmannosamine to obtain 3-fluoroneuraminic acid.
 13. Anenzymatic synthesis according to claim 12, wherein said enzyme activityranges from 0.02 to 1.0 U/ml.
 14. An enzymatic synthesis according toclaim 8, wherein the product has two diastereomers, an F-axialdiastereomer and an F-equatorial diastereomer, and, at the conclusion ofthe synthesis and without purification, the percentage of the productwhich is said F-axial diastereomer is about 75 to about 100%.
 15. Anenzymatic synthesis according to claim 14, wherein said percentageranges from about 91 to about 98%.
 16. An enzyme synthesis according toclaim 1, wherein said amount of said fluorinated sugar, which comprisesaxial-F and equatorial-F diastereomers, is taken up in an ion exchangemedium and purified or separated into separate yields of axial-F andequatorial-F diastereomers.
 17. A method for studying physiology ordiagnosing disease states in mammals, comprising: (a) synthesizing afluorinated sugar having at least 8 carbon atoms by reacting, in areaction medium, a F—CH₂—CO—COO⁻M⁺ with a furanoside or pyranoside sugarsubstrate in the presence of an aldolase enzyme, wherein M⁺ is H⁺ or apharmaceutically acceptable cation, and wherein the concentration ofaldolase enzyme, per 50 mM of F—CH₂—CO—COO⁻M⁺, is 1 to 2,500 U*/ml,where U* represents an enzyme activity unit and is the enzyme activitywith respect to the standard reaction of pyruvate andN-acetylmannosamine to obtain the N-acetyl derivative of neuraminicacid; (b) administering the fluorinated sugar to a mammal; and (c)monitoring the biodistribution or the pharmacokinetics of saidfluorinated sugar in said mammal.
 18. A method according to claim 17,wherein the fluorinated sugar of said step (a) is administered to themammal without separating the fluorinated sugar from the reactionmedium.
 19. A method according to claim 17, wherein the fluorinatedsugar of said step (a) is isolated from the reaction medium before it isadministered to the mammal.
 20. An enzymatic synthesis of a fluorinatedsugar having at least 8 carbon atoms, said synthesis comprising reactinga F—CH₂—CO—COO⁻M⁺ with a mannose substrate in the presence of analdolase enzyme, wherein M⁺ is H⁺ or a pharmaceutically acceptablecation, and wherein the concentration of aldolase enzyme, per 50 mM ofF—CH₂—CO—COO⁻M⁺, is 1 to 2,500 U*/ml, where U* represents an enzymeactivity unit and is the enzyme activity with respect to the standardreaction of pyruvate and N-acetylmannosamine to obtain the N-acetylderivative of neuraminic acid.
 21. An enzymatic synthesis of afluorinated sugar having at least 8 carbon atoms, said synthesiscomprising reacting a F—CH₂—CO—COO⁻M⁺ with a furanoside or pyranosidesugar substrate in the presence of an aldolase enzyme, wherein M⁺ is H⁺or a pharmaceutically acceptable cation, wherein the synthesis is abatch process wherein active aldolase enzyme is recovered and reused insubsequent batches, and wherein the concentration of aldolase enzyme,per 50 mM of F—CH₂—CO—COO⁻M⁺, is 1 to 2,500 U*/ml, where U* representsan enzyme activity unit and is the enzyme activity with respect to thestandard reaction of pyruvate and N-acetylmannosamine to obtain theN-acetyl derivative of neuraminic acid.
 22. An enzymatic synthesis of afluorinated sugar having at least 8 carbon atoms, said synthesiscomprising reacting a F—CH₂—CO—COO⁻M⁺ with a furanoside or pyranosidesugar substrate in the presence of an aldolase enzyme, wherein M⁺ is H⁺or a pharmaceutically acceptable cation, wherein said amount of saidfluorinated sugar, which comprises axial-F and equatorial-Fdiastereomers, is taken up in an ion exchange medium and purified orseparated into separate yields of axial-F and equatorial-Fdiastereomers, and wherein the concentration of aldolase enzyme, per 50mM of F—CH₂—CO—COO⁻M⁺, is 1 to 2,500 U*/ml, where U* represents anenzyme activity unit and is the enzyme activity with respect to thestandard reaction of pyruvate and N-acetylmannosamine to obtain theN-acetyl derivative of neuraminic acid.